1. Field of the Invention
The present invention relates generally to transgenic maize plants which are resistant to the herbicides and methods of using same. More specifically, it relates to the maize transformation events GA21, GG25, FI117 and GJ11.
2. Description of the Related Art
Chemical weed control is a powerful tool of our technological age. Long known as one of the most arduous of agricultural operations, weed killing has taken on an entirely new aspect as chemical after chemical is added to the arsenal of herbicides. The U.S. has led the world both in production and use of herbicides and as a result yields of maize, soybeans, cotton, sugar beets, and many other crops have increased since 1945, in some cases 100% or more. Thus while use of fertilizers and new high-yielding crop varieties have contributed greatly to the xe2x80x9cgreen revolutionxe2x80x9d chemical weed control has been at the forefront in technological achievement.
A particularly useful type of herbicide is one having a broad spectrum of herbicidal activity. Use of such herbicides obviates the need for application of multiple herbicides. The problem with such herbicides is that they typically have a deleterious effect on any crops which are exposed to the herbicide. One way to overcome this is to produce transformed crop plants with genes which confer resistance to certain broad spectrum herbicides.
Recent advances in genetic engineering have provided the requisite tools to transform plants to contain foreign genes. Plants may, therefore, be produced which have unique characteristics of agronomic importance. Certainly, weed control via herbicide tolerance is one such advantageous trait which is highly cost effective and environmentally compatible. Herbicide-tolerant plants may reduce the need for tillage to control weeds, thereby effectively reducing soil erosion. Further, herbicide resistant plants can reduce the number of different herbicides applied in the field.
One herbicide which is the subject of much investigation in this regard is N-phosphonomethyl-glycine, commonly referred to as glyphosate. Glyphosate inhibits the shikimic acid pathway which leads to the biosynthesis of aromatic compounds including amino acids and vitamins. Specifically, glyphosate inhibits the conversion of phosphoenolpyruvic acid and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase or EPSPS).
It has been shown that glyphosate tolerant plants can be produced by introducing, into the genome of the plant, the capacity to produce a higher level of EPSP synthase which enzyme is preferably glyphosate tolerant (Shah et al., 1986). The introduction into plants of glyphosate degradation gene(s) can provide a means of conferring glyphosate tolerance to plants and/or to augment the tolerance of transgenic plants already expressing a glyphosate tolerant EPSP synthase depending upon the physiological effects of the degradation products.
Glyphosate metabolism (degradation) has been examined in a wide variety of plants and little degradation has been reported in most of those studies. In those instances where degradation has been reported, the initial breakdown product is usually aminomethylphosphonate (AMPA) (Coupland, 1985; Marshall et al., 1987). In these instances, it is not clear if glyphosate is metabolized by the plant or by the contaminating microbes on the leaf surface to which glyphosate was applied. AMPA has been reported to be much less phytotoxic than glyphosate for most plant species (Franz, 1985) but not for all plant species (Maier, 1983; Tanaka et al., 1986). Glyphosate degradation in soils is much more extensive and rapid (Torstensson, 1985). The principal breakdown product identified is AMPA (Rueppel et al., 1977; Nomura and Hilton. 1977); a phosphonate that can be metabolized by a wide variety of microorganisms (Zeleznick et al., 1963; Mastalerz et al., 1965; Cook et al., 1978; Daughton et al., 1979a; 1979b; 1979c; Wackett et al., 1987a). A number of pure cultures of bacteria have been identified that degrade glyphosate by one of the two known routes (Schowanek and Verstraete, 1990; Weidhase et al., 1990; Liu et al., 1991). A route involving a xe2x80x9cC-P lyasexe2x80x9d that degrades glyphosate to sarcosine and inorganic orthophosphate (Pi) has been reported for a Pseudomonas sp. (Shinabarger and Braymer, 1986; Kishore and Jacob, 1987) and an Arthrobacter sp. (Pipke et al, 1987b). Pure cultures capable of degrading glyphosate to AMPA have been reported for a Flavobacterium sp. (Balthazor and Hallas, 1986), for a Pseudomonas sp. (Jacob et al, 1988) and for Arthrobacter atrocyaneus (Pipke and Amrhein, 1988). In addition, a large number of isolates that convert glyphosate to AMPA have been identified from industrial activated sludges that treat glyphosate wastes (Hallas et al., 1988). However, the number and nature of bacterial genes responsible for these degradations have not been heretofore determined nor have the gene(s) been isolated.
The development of plants resistant to the herbicidal compound glyphosate has been a goal in the engineering of many plant species (U.S. Pat. No. 4,769, 061). The development of glyphosate resistant tobacco plants was reported by Comai et al., (1985). Herbicide resistance was conferred on plants by expression of an aroA gene derived from Salmonella typhimurium encoding a glyphosate resistant form of the enzyme EPSP synthase. In addition, glyphosate resistant soybeans were produced (Monsanto, APHIS petition 93-258-01p). Methods for production of glyphosate resistant corn plants also have been described (WO 95/06128; U.S. Pat. No. 5,554,798). Similarly, a glyphosate oxidoreductase gene has been described for use in conferring glyphosate resistance (U.S. Pat. No. 5,463,175).
The ultimate goal in producing transgenic glyphosate resistant maize plants is to provide plants which may be treated with glyphosate at a level sufficient for killing weeds, without a deleterious effect on yield or fertility. In this respect, the prior art has failed. There is, therefore, a great need in agriculture for maize plants which can be directly sprayed in the field with glyphosate, thereby killing weeds, but otherwise not producing a deleterious effect on the crop itself.
The present invention seeks to overcome deficiencies in the prior art by providing fertile transgenic maize plants which can be treated with glyphosate in the field without a resulting loss in yield or fertility. Therefore, one aspect of the present invention relates to a fertile transgenic maize plant comprising a chromosomally incorporated expression cassette. In particular embodiments the expression cassette comprises: (i) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106, and (ii) a promoter active in maize operably linked to said EPSPS gene, wherein the yield of said fertile transgenic maize plant is not affected by a glyphosate application rate that affects the yield of a maize plant lacking said modified maize gene.
In another aspect, the maize plant may comprise a promoter which is selected from the group consisting of a rice actin promoter, a maize histone promoter and a fused CaMV 35S-Arabidopsis histone promoter. In one embodiment, the plant may comprise an expression cassette which is derived from pDPG434, pDPG427 or pDPG443. The expression cassette may, in particular embodiments, be further be defined as pDPG434, and the maize plant may be further defined as comprising a transformation event selected from the group consisting of GA21 and FI117; seeds comprising these events having been deposited with the ATCC and assigned the ATCC accession numbers ATCC 209033, and ATCC 209031, respectively. The maize plant comprising the FI117 transformation event may further be defined as comprising a bar gene.
In yet another aspect, the maize plant may comprise a pDPG427 expression cassette and may be further defined as comprising the transformation event GG25 or, may comprise an expression cassette of pDPG443 and the maize plant may be further defined as comprising the transformation event GJ11; seeds comprising the GG25 and GJ11 transformation events having been deposited with the ATCC and assigned the ATCC accession numbers ATCC 209032 and ATCC 209030, respectively. The invention is intended to include the progeny of any generation and seeds of the above maize plants, as well as the seeds of the progeny of any generation.
Still yet another aspect of the current invention comprises a method of preparing a fertile transgenic maize plant. The method comprises: (i) providing an expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; (ii) contacting recipient maize cells with said expression cassette under conditions permitting the uptake of said expression cassette by said recipient cells; (iii) selecting recipient cells comprising a chromosomally incorporated expression cassette; (iv) regenerating plants from said selected cells; and (v) identifying a fertile transgenic maize plant, the yield of which is not affected by a glyphosate application rate that affects the yield of a maize lacking said modified maize gene.
The method may comprise any method of contacting including, but not limited to, microprojectile bombardment, electroporation, or Agrobacterium-mediated transformation. Said selecting may comprise treating recipient cells with glyphosate. The promoter may be selected from the group consisting of a rice actin promoter, a maize histone promoter and a fused CaMV 35S-Arabidopsis histone promoter. In particular embodiments, said expression cassette may be derived from pDPG434, pDPG427 and/or pDPG443. The expression cassette may, in particular, be pDPG434 and the maize plant may be further defined as comprising a transformation event selected from the group consisting of GA21 and FI117. In the method, the transformation event may also be FI117, and said maize plant may further defined as comprising a bar gene. The expression cassette may also be pDPG427, and the maize plant may be further defined as comprising the transformation event GG25. The method also includes an expression cassette of pDPG443 where the maize plant may be further defined as comprising the transformation event GJ11.
In still yet another aspect, the invention is a fertile transgenic maize plant prepared according to a method comprising: (i) providing an expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; (ii) contacting recipient maize cells with said expression cassette under conditions permitting the uptake of said expression cassette by said recipient cells; (iii) selecting recipient cells comprising a chromosomally incorporated expression cassette; (iv) regenerating plants from said selected cells; and (v) identifying a fertile transgenic maize, the yield of which is not affected by a glyphosate application rate that affects the yield of a maize lacking said modified maize gene. The maize may have a promoter selected from the group consisting of a rice actin promoter, a maize histone promoter and a fused CaMV 35S-Arabidopsis histone promoter. The expression cassette may be derived from pDPG434, pDPG427 and pDPG443. The invention includes progeny of any generation and seeds of the fertile transgenic maize plant, as well as seeds of the progeny of the maize plant.
Still yet another aspect of the current invention is a glyphosate resistant, inbred, fertile maize plant comprising a chromosomally incorporated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene. The promoter may be selected from the group consisting of a rice actin promoter, a maize histone promoter and a fused CaMV 35S-Arabidopsis histone promoter. The expression cassette may be derived from pDPG434, pDPG427 and pDPG443. In particular embodiments the inbred maize plant may be further defined as comprising a transformation event selected from the group consisting of GJ11, FI117, GG25 or GA21, seeds comprising these transformation events having been deposited and assigned the ATCC accession numbers ATCC 209030, ATCC 209031, ATCC 209032, and ATCC 209033, respectively.
Still yet another aspect of the current invention is a glyphosate resistant, crossed fertile transgenic maize plant prepared according to the method comprising: (i) obtaining a fertile transgenic maize plant comprising a chromosomally incorporated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; (ii) crossing said fertile transgenic maize plant with a second maize plant lacking said expression cassette to obtain a third maize plant comprising said expression cassette; and (iii) backcrossing said third maize plant to obtain a backcrossed fertile maize plant; wherein said modified EPSPS gene is inherited through a male parent. In particular embodiments the second maize plant is an inbred. The third maize plant may be a hybrid. The maize plant may, in particular embodiments be further defined as comprising a transformation event selected from the group consisting of GJ11, F117, GG25 or GA21, ATCC accession numbers ATCC 209030, ATCC 209031, ATCC 209032, and ATCC 209033, respectively.
Still yet another embodiment of the invention is a glyphosate resistant, crossed fertile transgenic maize plant prepared according to the method comprising: (i) obtaining a fertile transgenic maize plant comprising a chromosomally incorporated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; and (ii) crossing said fertile transgenic maize plant with a second maize plant lacking said expression cassette to obtain a third maize plant comprising said expression cassette; wherein said modified EPSPS gene is inherited through a female parent. In particular embodiments, the second maize plant may be an inbred, and the third maize plant may be a hybrid. The maize plant may, in particular embodiments, be further defined as comprising a transformation event selected from the group consisting of GJ11, FI117, GG25 or GA21, seeds comprising these transformation events having been deposited and assigned the ATCC accession numbers ATCC 209030, ATCC 209031, ATCC 209032, and ATCC 209033, respectively.
Still yet another aspect of the invention is a glyphosate resistant, crossed fertile transgenic maize plant prepared according to the method comprising: (i) obtaining a fertile transgenic maize plant comprising a chromosomally incorporated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; (ii) crossing said fertile transgenic maize plant with a second maize plant to obtain a third maize plant comprising said expression cassette; and (iii) backcrossing said third maize plant to obtain a backcrossed fertile maize plant; wherein said modified EPSPS gene is inherited through a female parent. In particular embodiments, the maize plant may be an inbred and the third maize plant may be a hybrid. In one embodiment the maize plant may be further defined as comprising a transformation event selected from the group consisting of a GJ11, FI117, GG25 or GA21 transformation event, seeds comprising these transformation events having the ATCC accession numbers ATCC 209030, ATCC 209031, ATCC 209032, and ATCC 209033, respectively.
Still yet another aspect of the current invention is a glyphosate resistant, hybrid maize plant comprising a chromosomally incorporated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene. In one embodiment, the promoter is selected from the group consisting of a rice actin promoter, a maize histone promoter and a fused CaMV 35S-Arabidopsis histone promoter and the expression cassette is derived from pDPG434, pDPG427 and pDPG443. The maize plant may, in particular embodiments, be further defined as comprising a transformation event selected from the group consisting of GA21, GG25, GJ11 and FI117.
Still yet another aspect of the invention is a glyphosate resistant, hybrid, transgenic maize plant prepared according to the method comprising crossing a first and second inbred maize plant, wherein one of said first and second inbred maize plants comprises a chromosomally incorporated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene. In one embodiment, the promoter is selected from the group consisting of a rice actin promoter, a maize histone promoter and a fused CaMV 35S-Arabidopsis histone promoter, and said expression cassette is derived from pDPG434, pDPG427 and/or pDPG443. The maize plant may, in particular embodiments, be further defined as comprising a transformation event selected from the group consisting of GA21, GG25, GJ11 and FI117.
Still yet another aspect of the invention is a glyphosate resistant, crossed fertile transgenic maize plant prepared by a process comprising: (i) obtaining a fertile transgenic maize plant comprising a chromosomally integrated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; (ii) crossing said fertile transgenic maize plant with a second maize plant to obtain a third maize plant comprising said expression cassette; and (iii) crossing said third fertile transgenic maize plant with a fourth maize plant to obtain a fifth transgenic maize plant comprising said expression cassette. In one embodiment, the second and fourth maize plants have the same genotype. In another embodiment the second and fourth maize plants have different genotypes.
Still yet another aspect of the invention is seed of a fertile, transgenic maize plant, said seed comprising a chromosomally incorporated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene, said seed prepared by a process comprising the steps of: (i) obtaining a parental fertile, transgenic maize plant comprising a chromosomally incorporated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; (ii) breeding said parental plant with a second fertile maize plant to produce a plurality of progeny fertile, transgenic maize plants, said progeny maize plants including plants that express a chromosomally incorporated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS product having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; (iii) selecting from said progeny maize plants a plant having resistance to glyphosate; and (iv) obtaining seed from said selected progeny maize plant. In one embodiment the progeny maize plants are two generations removed from the parental transgenic maize plant.
The progeny maize plants having resistance to glyphosate may be selected by testing plants for resistance to glyphosate at an application rate of, for example 1xc3x97, 2xc3x97, 3xc3x97or 4xc3x97(1xc3x97 is equivalent to 16 ounces of Roundup(trademark) per acre). In a particular embodiment, the second fertile maize plant is a non-transgenic maize plant and the plant is pollinated with pollen from a male parental transgenic maize plant. The parental maize plant may be pollinated with pollen from said second fertile maize plant and wherein said parental maize plant is a female parental transgenic maize plant.
Still yet another aspect of the invention is a method of increasing the yield of corn in a field comprising: (i) planting fertile transgenic maize plants transformed with an expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS protein having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; and (ii) applying glyphosate to said field at an application rate that inhibits the yield of a maize plant that does not comprise said modified maize gene, wherein the yield of said fertile transgenic maize plant is not affected by said glyphosate application. In particular embodiments, the glyphosate application rate may be 1xc3x97, 2xc3x97or 4xc3x97.
Still yet another aspect of the invention is a method of inhibiting weed growth in a corn field comprising: (i) planting fertile transgenic maize plants transformed with an expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS protein having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; and (ii) applying glyphosate to said field at an application rate that inhibits the yield of a maize plant that does not comprise said modified maize gene, wherein the yield of said fertile transgenic maize plant is not affected by said glyphosate application. In particular embodiments, the glyphosate application rate may be 1xc3x97, 2xc3x97, or 4xc3x97.
Still yet another aspect of the invention is a method of growing corn comprising: (i) planting fertile transgenic maize plants transformed with an expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS protein having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; and (ii) treating said corn with glyphosate at an application rate that inhibits the yield of a maize plant that does not comprise said modified maize gene, wherein the yield of said fertile transgenic maize plant is not affected by said glyphosate application. In particular embodiments, the application rate may be, 1xc3x97, 2xc3x97or 4xc3x97.
It is clear that the ability to provide even a single fertile, transgenic corn line is generally sufficient to allow the introduction of the transgenic component (e.g., recombinant DNA) of that line into a second corn line of choice. This is because by providing fertile, transgenic offspring, the practice of the invention allows one to subsequently, through a series of breeding manipulations, move a selected gene from one corn line into an entirely different corn line. Therefore, the current invention is intended to include any maize plant, from any generation, which has one or more transgenes comprising a GJ11, FI117, GG25 or GA21 transformation event; seeds comprising these transformation events having the ATCC accession numbers ATCC 209030, ATCC 209031, ATCC 209032, and ATCC 209033, respectively. The invention further includes the seeds of maize plants of any generation comprising the GJ11, FI117, GG25 or GA21 transformation events.
Still yet aspect of the invention is a method for producing animal feed. This method may include the steps of (i) obtaining a fertile transgenic maize plant comprising a chromosomally integrated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS protein having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to the EPSPS gene; (ii) cultivating the transgenic Zea mays plant; (iii) obtaining seed from the cultivated Zea mays plant; and (iv) preparing animal feed from said seed. In particular embodiments, the fertile transgenic maize plants are further defined as comprising DNA from a plasmid selected from the group consisting of pDPG434, pDPG427 and pDPG443. In further embodiments, the fertile transgenic maize plants will comprise a transformation event selected from the group consisting of: GJ11, GG25, FI117 and GA21.
Still yet another aspect of the current invention is a method for producing food comprising the steps of: (i) obtaining a fertile transgenic Zea mays plant comprising heterologous DNA comprising a transformation event selected from the group consisting of GG25, GJ11, FI117 and GA21, wherein the DNA is heritable; (ii) cultivating the transgenic Zea mays plant; (iii) obtaining seed from the cultivated Zea mays plant; and (iv) preparing human food from the seed. Also included in the current invention is a method for producing oil comprising: (i) obtaining a fertile transgenic Zea mays plant comprising heterologous DNA comprising a transformation event selected from the group consisting of GG25, GJ11, FI117 and GA21, wherein the DNA is heritable; (ii) cultivating the transgenic Zea mays plant; (iii) obtaining seed from the cultivated Zea mays plant; and (iv) preparing oil from the seed.
Still yet another aspect of the current invention is a method for producing starch comprising the steps: (i) obtaining a fertile transgenic Zea mays plant comprising heterologous DNA comprising a transformation event selected from the group consisting of GG25, GJ11, FI117 and GA21, wherein the DNA is heritable; (ii) cultivating said transgenic Zea mays plant; (iii) obtaining seed from the cultivated Zea mays plant; and (iv) preparing starch from the seed.
Still yet another aspect of the current invention is a method for producing seed comprising: (i) obtaining a fertile transgenic maize plant comprising a chromosomally integrated expression cassette comprising (a) a modified maize EPSPS gene encoding an EPSPS protein having isoleucine at position 102 and serine at position 106 and (b) a promoter active in maize operably linked to said EPSPS gene; (ii) cultivating said transgenic Zea mays plant; and (iii) obtaining seed from said cultivated Zea mays plant.
Still yet another aspect of the current invention provides a method of plant breeding comprising the steps of: (i) planting in pollinating proximity seeds capable of growing into first and second parent plants, wherein the first parent plant comprises a first transgene, the plant being able to be rendered male-sterile by treatment with a preselected herbicide, and wherein the first plant is resistant to said preselected herbicide; (ii) cultivating the seeds to produce the first and second parent plants; (iii) inducing male-sterility in the first parent plant by treating the plant with the preselected herbicide; (iv) allowing the second corn plant to pollinate the first parent plant; and (v) collecting seeds produced on the first plant. In particular embodiments the second parent plant is further defined as being resistant to the preselected herbicide.
The first and second plants may be selected from the group consisting of maize, wheat, rice, oat, barley, sorghum, sunflower, alfalfa and soybean. The preselected herbicide may be glyphosate, however, in other embodiments the herbicide may be glufosinate, imidazolinone, sulphonylurea, kanamycin, G418, bromoxynil or methotrexate. The first transgene may comprise a GG25 transformation event and/or a GJ11 transformation event, or any other suitable, similar transgene. The second plant may comprise a GA21 transformation event and/or a FI117 transformation event, or any other suitable, similar transgene. In particular embodiments the step of inducing male-sterility comprises applying a concentration of glyphosate of from 8 ounces per acre to 96 ounces per acre, which may be applied between the V5 and VT stages of development.
Still yet another aspect of the current invention is a method of testing seed quality of a hybrid maize seed comprising a herbicide resistance transformation event, such as GA21, GG25, FI117 or GJ11. The method comprises the steps of: (i) planting said seed; (ii) cultivating the seed; and (iii) treating the plants grown from the seed with a preselected herbicide. In particular embodiments the seeds are selected from the group consisting of maize seeds, wheat seeds, rice seeds, oat seeds, barley seeds, sorghum seeds, sunflower seeds, alfalfa seeds and soybean seeds. In other embodiments the seeds are maize seeds. The transformation event may comprise a mutated EPSPS and the preselected herbicide may be glyphosate. More specifically, the plants may be treated with from 8 to 96 ounces per acre of glyphosate, and this treatment may take place between the V4 and VT stages of development. Alternatively the gene may be another suitable herbicide resistance gene and the preselected herbicide selected from the group consisting of glufosinate, imidazolinone, sulphonylurea, kanamycin, G418, bromoxynil and methotrexate.
Still yet another aspect of the invention is a method of plant breeding comprising the steps: (i) planting a seed capable of growing into a first plant, the plant comprising a transformation event conferring herbicide resistance; (ii) cultivating the seed to produce the first plant; (iii) treating the first plant with a preselected herbicide to render pollen not having the transformation event inviable; (iv) allowing pollen having the transformation event to pollinate the first plant or a second plant, wherein the pollen having the transformation event remains viable following the treating; and (v) collecting seed from the first or the second plant. The transformation event may comprise a mutated EPSPS gene operably linked to a promoter functional in said first plant, and may further be a GA21 or FI117. Treating the first maize plant may comprise treating the first maize plant with from 8 to 96 ounces per acre of glyphosate, and may take place between the V4 and VT stages of development. The first plant may be selected from the group consisting of maize, wheat, rice, oat, barley, sorghum sunflower, alfalfa, and soybean. In addition to glyphosate, the preselected herbicide may also be selected from the group consisting of glufosinate, imidazolinone, sulphonylurea, kanamycin, G418, bromoxynil and methotrexate.